In project 3 of this P01, we focus on the development and execution of micro titer plate based enzyme assays for cell wall synthetic enzymes that are required by M. tuberculosis for viability. In the over all P01 a refinement cycle has been developed in which compounds are synthesized by a compound development module and analyzed by a compound analysis module. The biological activities of the compounds are reported to the compound development module and the cycle repeated until leads that are effective against MDR-TB and also effective against TB in mice are produced. One of our tasks in project 3 is to determine IC50's and, as appropriate, Ki's of inhibitors of "cycle ready enzymes" made by the compound development module. At present, these "cycle ready enzymes" include two enzymes involved in the synthesis of dTDP-rhamnose (RmlC and RmlD) and the enzyme UDP-galactopyranose mutase (GIf). By "cycle ready enzyme targets" we mean that they have already had the assays developed for them, crystal structures determined, and inhibitors that can be used as starting points identified by screening chemical libraries. Another major task of project 3 is to develop four additional enzymes so that they can also be targeted by our refinement cycle (i.e. become "cycle ready"). These enzymes are GImU which synthesizes UDP-GIcNAc via two separate reactions, farnesyl diphosphate synthetase, decaprenyl diphosphate synthetase, and various galactofuranosyl transferases. For these enzymes microtiter plate based assays will be developed and chemical libraries screened to identify initial hits. The compound development module will then use this hits to form co-crystals with the enzymes and design and synthesize increasingly more active inhibitors in the context of the refinement cycle.